SEMINAR ON GERMPLASM CONSERVATION

Plant Cryopreservation at the huntington botanical gardensRaquel Folgado Casado

Cryopreservation Research Fellow

NEED FOR PLANT CONSERVATIONRare and threatened species

Plant breeding

Loss of genetic diversity among crop plant species. Human dependence on plant species for food and many different uses. E.g. : Basic food crops, building materials, oils, lubricants, rubber and other latexes, resins, waxes, perfumes, dyes fibres and medicines. Species extinction and many others are threatened and endangered deforestation. Great diversity of plants is needed to keep the various natural ecosystems functioning stably interactions between species. Aesthetic value of natural ecosystems and the diversity of plant species.2What is Germplasm?Germplasm broadly refer to the hereditary material (total content of gene) transmitted to the offspring through germ cell

It is also used to describe a collection of genetic resources for an organism

germplasm conservationPlant germplasm (genetic source material for plant breeding)

They may include: SeedsOther plant propagules such as Leaf Stem Pollen Cultured cells

which can be grown into mature plant

CONSERVATION OF PLANT GERMPLAMIn situ:natural environment(-)High risks of losses, highly exposedEx situ: botanical gardens, research centres, seed and germplasm banksEx vitro:Traditional method (field collections)(-)Infection risks, labour-intensiveIn vitro:Vegetatively propagated speciesNormal growth Slow growth (temp, O2 , H2O , medium ~)

(+)Sterile material, no risks by insects or weather conditions(-)Still labour-intensive, risks for contamination,human error or somaclonal variationCryopreservation (-196C): Long-term germplasm conservation(+)Unlimited storage time, reduced costs & space,prevention of infection and genetic changesWhat is cryopreservation?Cryopreservation (Greek, krayos-frost) literally mean in the frozen stateProcess of cooling, or freezing, and storing cells, tissues or organs to ultra-low temperatures and keep them for a future use

USE OF CRYOPRESERVATIONPlant materials (cell/tissue) of several species can be cryopreserved and maintained for several years, and used as and when needed. Cryopreservation is an ideal method for long term conservation of cell culture which produce secondary metabolites e.g. medicines Disease (pathogen) free plant material can be frozen and propagated whenever required. Recalcitrant seeds can be maintained for long . Conservation of somaclonal and gametoclonal variation in culture. Plant material from endangered species can be conserved. Cryopreservation is a good method for the selection of cold resistant mutant cell lines which could develop into frost resistant plant .

7In vitro PLANT MATERIAL FOR CRYOPRESERVATION

Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperm, ovules, seeds, culture plants cells, protoplast, callus. In the picture we present the different in vitro plant material that can be cryopreservedIn vitro method employing shoots, meristems and embryos are ideally suited for the conservation of germplasm. The plant with recalcitrant seeds and genetically engineered can also be preserved by this in vitro approach.There are several advantages associated with in vitro germplasm conservationLarge quantities of material can be preserved in small space The germplasm preserved can be maintained in an environment free from pathogens.It can be protected against the natures hazardsFrom the germplasm stock large number of plants can be obtained whenever needed.

8Mechanism of cryopreservationKey: avoidance of the formation of intracellular ice

Presence of salts and organic molecules in the cells, the cell water requires much more lower temperature to freeze (even up to -68C)

At ultra-low temperature (such as -196C), the metabolic processes and biological deteriorations in the cells/tissues are stopped.The technique of freeze preservation is based on the transfer of water present in the cells from a liquid to solid state. Due to the presence of salts and organic molecules in the cells, the cell water requires much more lower temperature to freeze (even up to -68C) compared to the freezing point of pure water (around 0C) . When stored at low temperature , the metabolic processes and biological deteriorations in the cells/tissues almost come to standstill.

9Technique of cryopreservationThe cryopreservation of plant material involves the following stagesDevelopment of sterile tissue culture.Addition of cryoprotectant and pretreatmentFreezing StorageThawingRecultureMeasurement of survival/viabilityPlant regeneration

Development of sterile tissue cultureMorphological and physiological characters largely influence the ability of the explants to survive in cryopreservation.

The selection of plant species and the tissue with particular reference to the morphological and physiological characters largely influence the ability of the explants to survive in cryopreservation . Any tissue from a plant can be used for cryopreservation e.g. meristems, embryos, endosperm, ovules, seeds, culture plants cells, protoplast, callus.

11Addition of cryoprotectant Prevent the damage caused to cells by freezing or thawingDimethyl Sulfoxide (DMSO)GlycerolEthylenePropyleneSucroseMannoseGlucoseProlineAcetamide Freezing Slow freezing method : at 0.5-5C/min from 0C to -100C, and then transferred to LNRapid freezing method :-300 to -1000C/minStepwise freezing method: Intermediate temperature for 30 min., and rapidly coolDry freezing method :Dehydration of material before freezing

he sensitivity of the cell to low temperature is variable and largely depends on the plant species. Four different types of freezing method are used:

13Storage

Range of -72 to -196C Over solid carbon dioxide (at -79C) Low temperature deep freezers (at -80C) In liquid nitrogen (at -196C, gas phase -150C)

The ultimate objective of storage is to stop all the cellular metabolic activities and maintain their viability

For long term storage, temperature at -196C in liquid nitrogen is ideal.

Thawing Warm water (temp 37 45C) bath with vigorous swirling

Rapid thawing (at the rate of 500-750C min) occurs, and this protects the cells from the damaging effects ice crystal formation.

Reculture and plant regeneration

Cryoprotectant is removed and plant material is cultured

Growth promoting and maintenance of appropriate environmental conditions

Ultimate purpose: regenerate desired plantIn general thawned germplasm is washed several times to remove cryoprotectant. The material is then recultured in a fresh mediaThe ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant . For appropriate plant growth and regeneration , the cryopreserved cell/tissues have to be carefully nursed, grown. Addition of certain growth promoting substances, besides maintenance of appropriate environmental conditions is often necessary for successful plant regeneration

16Limitations of cryopreservationThe expensive equipment needed to provide controlled and variable rates of cooling/warming temperatures

Formation of ice crystals inside the cell should be prevented

Sometimes certain solutes from the cell leak out during freezing

Cryoprotectant also affect the viability of cells

CRYOPRESERVATION AT HBGSUCCULENTS AND CACTIAgave and Aloe: optimization of the plant regeneration process

The tests with cacti seed are also promising and therefore, we will be able to store some species into liquid nitrogen

AVOCADO (Persea americana)

7 Varieties in TC:

Mexicola (M)Nabal (G)Nimlioh (G)Carlsbad (G)Anaheim (G)Zutano (Hybrid M x G)Mayo (Hybrid M x G)Cryopreservation of meristems (collaboration with group at University of Queensland, Australia)

MAGNOLIA spp

12 species introduced in TC:

M. grandifloraM. macrophylla spp dealbataM. macrophylla spp asheiM. champacaM. pacificaM. doltsopaM. aromaticaM. dawsonianaM. denudataM. acuminata var SubcordataM. ernestiiM. x soulangeanaM. tripetala

ORCHIDSCryopreservation of seedsClones of interest from Huntington living collection

CAMELLIA

Introduction to TC:C. hiemalis var.KanjiroC. japonicaC. hongconensisC. grosiiTITAN ARUM (Amorphophallus titanum)

Pollen collected and freezedSeeds collected and tested for cryopreservation

OSMOTIC STRESS TO IMPROVE CRYOPRESERVATION TOLERANCE

In summaryThe Huntington might be a Botanical Garden of reference in America for the long-term conservationWe intend to provide new tools for plant conservation to other institutions and usersCryopreservation technologies may be crucial to the conservation of plant diversity worldwide

Thanks!!